DESCRIPTION: (Applicant's Abstract) The ryanodine receptor (Ryr, Ca2+ release channel) plays a central role in muscle contraction. It is mainly localized where the sarcoplasmic reticulum (SR) forms junctions with T-tubular and surface membranes (T-SR junction). It is responsible for release of Ca2+ from SR that triggers contraction. Release channels are suggested to be activated by two different mechanisms: mechanical interaction with dihydropyridine receptor (DHPr, voltage sensor) and calcium-induced calcium release (CICR). Skeletal muscle contains different Ryr isoforms that might contribute to the diversity of excitation-contraction coupling (ECC) mechanisms. My preliminary data disclosed two distinctive types of local Ca2+ signaling: Ca2+ sparks and release in small events. The goal of this proposal is to investigate the role of different release channel isoforms in Ca2+ release. I propose to take advantage of the fact that relative amounts of two major skeletal muscle Ryr isoforms (Ryrl and Ryr3) vary widely between muscle types and during muscle development in order to test three major hypotheses: 1. Ca2+ release in small events is directly controlled by voltage and provides a precursor Ca2+ that activates Ca2+ sparks. 2. Ca2+ sparks are produced by opening of multiple Ryr Ca2+ release channels. 3. Ryr3rs are the channels required to produce sparks in skeletal muscle and are activated by CICR. For this we will explore local calcium signaling in a variety of mature and developing mammalian, amphibian and fish skeletal muscle fibers with different Ryr isoforms composition and/or different numbers of Ryrs per T-SR junction. In parallel, we will monitor tissue expression of different Ryr isoforms by immunoblotting and/or immunostaining. Some muscle illnesses are increasingly been understood in terms of defects of molecules participating in ECC. Proposed studies will greatly advanced our knowledge of their functioning, which in turn could improve our understanding of their role in decease.